CN113171448A

CN113171448A - Prostate cancer nucleic acid vaccine

Info

Publication number: CN113171448A
Application number: CN202110399431.XA
Authority: CN
Inventors: 于继云; 王宇; 李忠明
Original assignee: Gu'an Dingtai Haigui Biological Technology Co ltd
Current assignee: Beijing Zhendan Dingtai Biotechnology Co ltd
Priority date: 2015-11-11
Filing date: 2015-11-11
Publication date: 2021-07-27
Also published as: CN105343874A

Abstract

The invention discloses a prostate cancer nucleic acid vaccine. The nucleic acid vaccine is formed by fusing the region which covers HLA-A2 epitope and can induce CD8+ T cell immune response of prostate cancer antigens PSCA, PAP, PSMA and PSA, wherein the upstream fusion expresses a signal peptide and an extracellular region of FLT3L, and the downstream fusion expresses CD 40L. The invention carries out fusion expression on 4 kinds of prostate cancer antigens, and is assisted by various costimulatory molecules and intramolecular adjuvants, thereby improving the immunogenicity of the nucleic acid vaccine and enhancing the anti-tumor immune effect of the nucleic acid vaccine. The experimental result shows that the nucleic acid vaccine with the fusion expression mode can induce antibody reaction and cellular immunity simultaneously in immunized mice, and the cellular immunity reaction is particularly strong.

Description

Prostate cancer nucleic acid vaccine

This application is filed as a divisional application on application date 2015, 11/11, application number 201510765091.2, entitled "a prostate cancer nucleic acid vaccine".

Technical Field

The invention relates to a nucleic acid vaccine, in particular to a prostate cancer nucleic acid vaccine.

Background

The incidence of prostate cancer is on the rise worldwide and becomes a major threat to human health. The prostate cancer without metastasis can effectively inhibit tumor development through chemotherapy, radiotherapy and other treatments, and the patient can obtain more ideal survival rate. However, most of recurrent and metastatic prostate cancer will develop into castration-resistant prostate cancer (CRPC) after traditional treatment, and at this time, the existing treatment means cannot effectively control the tumor development, so the survival rate of the patient is very low. Therefore, there is a need for a new and effective therapeutic approach to treat CRPC.

Over the last decade, immunotherapy has received increasing attention as a new approach to tumor therapy. Because of its unique advantages, anti-tumor DNA vaccines have been developed rapidly, and many DNA vaccines against human tumors have entered clinical trials, some of which have been approved for the treatment of animal tumors. The research on immunotherapy methods for prostate cancer is relatively slow, and prostate cancer expresses various Tumor Associated Antigens (TAAs), such as prostate specific antigen PSA, prostate specific membrane antigen PSMA, prostate acid phosphatase PAP, prostate stem cell antigen PSCA, and the like. These tumor-associated antigens have the potential to provide multiple targets for immunotherapy. In addition, the serum PSA concentration of prostate cancer patients changes at an early stage, which makes early treatment possible. At the same time, T cells reacting with prostate TAA could be detected in the circulating blood, indicating that self-tolerance to these antigens could be overcome. The key goal to be achieved with tumor DNA vaccines is to elicit a cell-mediated immune response in the body, primarily by activating Tumor Associated Antigen (TAA) -specific killer T lymphocytes (CTLs), which then achieve therapeutic effects by killing cells expressing the Tumor Associated Antigen (TAA). This means that it would be possible to fight castration-resistant prostate cancer if the DNA vaccine could elicit a sufficiently strong immune response in the body. The selection of antigen is particularly important to make DNA vaccines have a good effect.

The prostate specific antigens PSCA, PAP, PSMA, PSA all contain good targets (epitopes) for immunotherapy of prostate cancer. However, the traditional Chinese medicine composition has the advantages and the disadvantages, can play a certain role when being used alone, and has no obvious effect. Patent document CN1696293A (hereinafter referred to as document 1) discloses a genetic recombinant chemotactic antigen vaccine, which relates to a genetic recombinant prostate cancer vaccine, and the nucleotide sequence of the genetic recombinant prostate cancer vaccine is an artificially spliced SLC-PSM-mPAP-PSA-Fc (SLC-3P-Fc for short). However, the genetic recombinant prostate cancer vaccine provided in this document 1 must rely on the synergistic effect of the Fc fragments of the chemokines SLC and IgG fused upstream and downstream, respectively, to induce specific anti-tumor cellular and humoral immune responses, while the nucleic acid vaccine (psig-3P) fused with only the expression plasmid of the chemokine SLC secretory peptide gene upstream of the 3P expression gene is completely unable to induce specific anti-tumor responses (see fig. 13 and 14 of document 1), indicating that the specific anti-tumor response effect of the prostate cancer specific antigens PSM, PAP, and PSA after fusion (3P) is also insignificant. The specific functional epitopes contained in PSCA, PAP, PSMA, PSA and the expression effect of fusion (4P) thereof are not known at present.

Nowadays, prostate cancer DNA vaccines have entered into clinical trial stage from laboratory experiment and have made some progress, however, the clinical effect of the current prostate cancer DNA vaccines is not obvious, even there is only theoretical effect, and there is no practical effect.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention prepares the prostate cancer nucleic acid vaccine by fusing gene sequences expressing prostate cancer antigens PSCA, PAP, PSMA and PSA related epitopes.

A prostate cancer nucleic acid vaccine is formed by fusing gene sequences of a region covering HLA-A2 epitope for expressing prostate cancer antigens PSCA, PAP, PSMA and PSA and capable of inducing CD8+ T cell immune response, wherein a signal peptide for expressing FLT3L and a gene sequence of an extracellular region are fused at the upstream of the nucleic acid vaccine, and a gene sequence for expressing CD40L is fused at the downstream of the nucleic acid vaccine;

the amino acid sequence of the region which can induce CD8+ T cell immune response and covers HLA-A2 epitope of the prostate cancer antigen PAP is shown as SEQ ID NO. 16;

the amino acid sequence of the region which covers HLA-A2 epitope and can induce CD8+ T cell immune response of the prostate cancer antigen PSA is shown as SEQ ID NO. 17;

the amino acid sequence of the region covering HLA-A2 epitope of the prostate cancer antigen PSCA capable of inducing CD8+ T cell immune response is shown as SEQ ID NO. 18;

the amino acid sequence of the region covering HLA-A2 epitope of the prostate cancer antigen PSMA capable of inducing CD8+ T cell immune response is shown as SEQ ID NO. 19.

The gene sequence of the prostate cancer nucleic acid vaccine is shown in a nucleotide sequence table SEQ ID NO 1.

A plasmid vector comprising the prostate cancer nucleic acid vaccine described above.

The original plasmid vector of the plasmid vector was pSVK 1.

Eukaryotic expression cells comprising the plasmid vectors described above.

The original cell of the eukaryotic expression cell is 293T cell.

The application of the prostate cancer nucleic acid vaccine in preparing the anti-prostate cancer medicament.

The medicine comprises the prostate cancer nucleic acid vaccine and a co-stimulation adjuvant, wherein the co-stimulation adjuvant comprises cytokines, chemokines and bacterial toxins.

Preferably, the cytokines are the cytokines GM-CSF and B7.1.

Compared with the prior art, the invention has the following beneficial effects: the invention designs a novel nucleic acid vaccine which is obtained by fusion expression of 4 common antigens of prostate cancer, and is supplemented with various co-stimulatory molecules and intramolecular adjuvants, so that the immunogenicity of the nucleic acid vaccine is improved, and the anti-tumor immune effect of the nucleic acid vaccine is enhanced. According to the nucleic acid vaccine with the fusion expression mode, the immunized mice can simultaneously induce antibody response and cellular immunity, and the data show that the immune response induced after the immunization of the vaccine group is very strong, especially the strong cellular immune response can be induced, which is very important for the anti-tumor effect of the vaccine.

Drawings

FIG. 1 is a diagram showing the double restriction enzyme identification of pUC57-FL +4P + CD40L plasmid;

in the figure, M: 5000DNA marke; 1: pUC57-FL +4P + CD40L plasmid; 2: plasmid BamH I and Mfe I of pUC57-FL +4P + CD40L were digested simultaneously.

FIG. 2 is a drawing of the linearized identification of pVAX-GB plasmid;

in the figure, M: 5000DNA marke; 1: pVAX-GB plasmid; 2: the pVAX-GB plasmid BamH I and EcoR I were digested simultaneously.

FIG. 3 is the restriction identification of the transition vaccine plasmid;

in the figure, M: 15000DNA marker; 1: a vaccine plasmid; 2: the vaccine plasmid Pme I + BamH I was digested simultaneously.

FIG. 4 flow cytometric analysis of transient expression of vaccine plasmids in 293T cells;

in the figure, A: control group (FITC + PE); b: experimental group (CD154 PE + CD80 FITC).

FIG. 5 shows the results of immunohistochemistry of muscle tissues of mice immunized with vaccine plasmids;

in the figure, A: saline group mice; b: vaccine group mice (PSCA); c: vaccine group mice (PAP); d: vaccine group mice (PSA); e: vaccine group mice (PSMA).

FIG. 6 shows the values of specific antibody OD450nm in the serum of each group of mice measured by ELISA.

FIG. 7 is an ELISPOT spot diagram.

FIG. 8 shows the results of the ELISPOT assay of IFN-. gamma.in mice after immunization.

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

Example 1 design of vaccine fusion antigen and construction and preparation of expression plasmid

Experimental materials:

plasmid pVAX-IRES- (GM-CSF + B7) containing adjuvant molecules GM-CSF (Genbank No. M11220.1), B7.1(Genbank No. NM-005191.3) and internal ribosome entry site IRES sequences is constructed as a prophase plasmid as shown in a sequence table SEQ ID NO:20, (pVAX-GB for short) and is used as a transition vector in the vaccine construction process (the vector is constructed by fusing IRES, GM-CSF and B7.1 sequences and then carrying out whole gene synthesis, and cloning the fused gene into pVAX1 of Invitrogen through BamH I and EcoR I enzyme cleavage sites); the empty vector pSVK1 for vaccine construction is constructed and stored in a previous stage (the vector is modified on the basis of pSFV1 plasmid of Invitrogen company, specifically, a CMV promoter is used for replacing an SP6 promoter, kanamycin resistance is used for replacing the original ampicillin resistance, and the whole sequence of the modified vector is shown as a sequence table SEQ ID NO: 21); coli DH 5. alpha. was stored as this company.

The ligase kit solution I is a product of Takara company; DNA restriction enzymes were purchased from NEB; the fragment glue recovery kit and the plasmid extraction kit are purchased from Kangkang, a century Biotechnology Co., Ltd; agarose for DNA electrophoresis was purchased from Promega corporation; the transfection reagent EntransTM-H is a product of Beijing Engyen Biotechnology GmbH; the PE-labeled anti-human CD154(CD40L) monoclonal antibody and the FITC-labeled anti-human CD80(B7-1) monoclonal antibody are products of Santa Cruz company;

experimental methods and results:

obtaining of FL-4P-CD40L fusion Gene

Four prostate cancer antigens were selected by consulting the literature: PSCA, PAP, PSMA and PSA, and further determines the region covering HLA-A2 epitopes of the four antigens capable of inducing CD8+ T cell immune response, 10 epitopes are selected (table 1), and a 4P antigen is designed according to the spatial positioning of natural proteins of the four antigens in cells when designing target antigens (table 2).

TABLE 1 regions covering the HLA-A2 epitope

Design of the Table 24P antigen

Meanwhile, in order to enhance the immunogenicity of the vaccine, the upstream fusion of the 4P antigen expresses the signal peptide and extracellular region of FLT3L, the downstream fusion of the 4P antigen expresses CD40L, the complete sequence of the fusion gene is synthesized by Jinzhi corporation, and the complete sequence is shown in a sequence table SEQ ID NO: 1.

2. Obtaining FL-4P-CD40L fusion antigen gene segment

Plasmid pUC57-FL +4P + CD40L synthesized by GenBank company is double cut with BamH I and Mfe I, and 2.3kb fragment is recovered by cutting gel, thus obtaining FL-4P-CD40L fusion gene, and the double-enzyme cutting identification result is shown in FIG. 1.

3. Double enzyme digestion linearization of intermediate transition DNA vector pVAX-GB

The plasmid pVAX-GB is subjected to double cutting by using BamH I and EcoR I to linearize the vector, then the vector is cut into gel and recovered, and the figure of enzyme cutting identification is shown in figure 1.

4. Construction and enzyme digestion identification of transition vaccine plasmid pVAX-FL-4P-CD40L-IRES-GM-CSF + B7 (transition vaccine plasmid for short)

And (3) connection reaction: mu.l of solution I was added to a 10. mu.l reaction system, and 4. mu.l of the obtained FL-4P-CD40L fragment and 1. mu.l of the obtained linearized pVAX-GB fragment were reacted at 16 ℃ for 30 min.

And (3) conversion of a connecting product: and (3) putting 100 mu l of competent cells in an ice bath, adding 10 mu l of the ligation product into the competent cell suspension after the competent cells are melted, gently mixing uniformly, carrying out ice bath for 30min, immediately transferring to a 42 ℃ water bath kettle, carrying out hot shock for 60s, and quickly transferring to the ice bath for 2 min. To the competent cells, 500. mu.l of sterile LB medium without antibiotics was added, mixed well and cultured at 37 ℃ for 40min with shaking at 200 rpm. Mu.l of the transformed competent cells were applied uniformly to a solid medium containing ampicillin (100. mu.g/mL), and cultured in an inverted state at 37 ℃ for 12 to 16 hours. About 6 single colonies were picked from the plate, inoculated in LB liquid medium (5 mL/tube) containing ampicillin (100. mu.g/mL), and cultured overnight at 37 ℃ with shaking at 200 rpm.

The recombinant plasmid was extracted according to the instructions of the plasmid extraction kit (Kangji Biotech Co., Ltd.), and the transient vaccine plasmid pVAX-FL-4P-CD40L-IRES-GM-CSF + B7 was identified by double-cutting with BamHI and PmeI, as shown in FIG. 3, and the gel electrophoresis yielded target fragments with expected sizes, about 4200bp and 3000 bp.

5. Construction and identification of vaccine plasmid pSVK1-FL-4P-CD40L-IRES-GM-CSF + B7 (hereinafter referred to as vaccine plasmid)

Enzyme digestion of empty vector pSVK 1: pSVK1 was digested with Sma I and plasmid pSVK1 was linearized, which now had two blunt ends after digestion.

Obtaining FL-4P-CD40L-IRES-GM-CSF + B7 fusion gene fragment: firstly, carrying out single enzyme digestion on a transition vaccine plasmid pVAX-FL-4P-CD40L-IRES-GM-CSF + B7 by using BamH I, purifying and recovering the plasmid and then filling the plasmid, then carrying out single enzyme digestion on the vector obtained in the previous step by using Pme I, and obtaining the FL-4P-CD40L-IRES-GM-CSF + B7 fusion gene fragment with two flat ends through the operation of the step.

Connection and identification of vector pSVK1 and FL-4P-CD40L-IRES-GM-CSF + B7 fusion gene:

the pSVK1 vector FL-4P-CD40L-IRES-GM-CSF + B7 fusion genes with two blunt ends obtained in the above steps were ligated, transformed E.coli.DH5. alpha. competence, cultured overnight on LB plate containing 30. mu.g/mL kanamycin resistance, and picked up as a single clone for amplification culture in LB liquid medium.

Because the connection of two flat ends has the problem of positive and negative directionality, a pair of primers is designed to identify the directionality of connection, and the primers are respectively selected from a vector and a fusion gene fragment. The positive clone screened by the PCR method of the bacterial liquid is the plasmid pSVK1-FL-4P-CD40L-IRES-GM-CSF + B7 (vaccine plasmid for short) of the nucleic acid vaccine for treating the prostate cancer.

6. Eukaryotic expression of vaccine plasmids

The vaccine plasmid is transiently transfected into 293T cells by a conventional experimental method, and then the expression of the vaccine plasmid in the 293T cells is detected by flow cytometry. 293T cells after 48h of transfection were collected, incubated with PE-labeled anti-human CD154(CD40L) monoclonal antibody and FITC-labeled anti-human CD80(B7-1) monoclonal antibody in the absence of light, and then detected by flow cytometry, the detection results of which are shown in FIG. 4.

Injecting vaccine plasmids into muscle tissues (quadriceps femoris) of BALB/c mice, stimulating by using an ECM830 type electric pulse introduction instrument, killing the immune mice by 36h posterior cervical dislocation, separating the muscle tissues of the immune parts of the mice by operation, and observing the expression condition of the exogenous genes of the muscle tissues of the injection parts of the recombinant plasmids through the steps of fixing, embedding, slicing, antibody incubation, dyeing and the like. After immunohistochemistry using rabbit anti-human PSCA, PAP, PSA, PSMA monoclonal antibodies, different amounts of tan particles were observed in muscle tissues of immunized mice, whereas no expression of the target protein was detected in muscle tissues of mice immunized with physiological saline (fig. 5). The results show that the fusion gene of the vaccine plasmid can be effectively expressed in a mouse body after intramuscular injection and electrical pulse stimulation.

Example 2 therapeutic prostate cancer nucleic acid vaccine immunogenicity assays

Experimental Material

Vaccine plasmid pSVK1-FL-4P-CD40L-IRES-GM-CSF + B7 was constructed as the first section of this section; competent Escherichia coli E.coli.DH5. alpha. was manufactured by this company; female BALB/c mice, 6-8 weeks old, were purchased from Experimental animals, Inc., Viton, Beijing. Synthetic peptide is a stimulator synthetic peptide used in ELISPOT and cytokine secretion experiments in immunological assays, and was completed by behcet bioscience, beijing. The kit for extracting endotoxin-removing plasmid in large quantity is purchased from Tiangen Biotechnology (Beijing) limited, the RPMI 1640 culture medium is a product of Gibco BRL company, the mouse lymphocyte separation liquid is purchased from Tianjin YangZhi biological product technology limited, the Murine IFN-gamma ELISPOT pre-coated kit is purchased from Shenzhen Dake company, and other reagents are domestic analytical purifications.

Experimental methods

1. Mass extraction and purification of plasmid DNA for immunization

The empty vector of the endotoxin-free plasmid pSVK1, the adjuvant plasmid pSVK1-GB and the vaccine plasmid pSVK1-FL-4P-CD40L-IRES-GM-CSF + B7 required by animal experiments are all operated according to the instruction of the endotoxin-free plasmid great improvement kit of Tiangen Biochemical technology (Beijing) Co., Ltd.

2. Grouping of laboratory animals

Female BALB/c mice 6-8 weeks old were randomly divided into 5 groups of 8 mice each. Group A is blank control group; group B is normal saline injection group; group C is pSVK1 empty vector group; group D is pSVK1-GB adjuvant group; group E is the pSVK1-FL-4P-CD40L-IRES-GM-CSF + B7 vaccine group.

3. Immunization strategy

The experimental mice are immunized with 3 times of vaccines every 7 days, the immunization is carried out by adopting a mode of injecting quadriceps femoris muscle and simultaneously carrying out electric pulse stimulation, the group A is a blank control group without injection, the group B is injected with 100 mu L of physiological saline, the group B is injected with 50 mu g/100 mu L/mouse of corresponding plasmids, C, D, E groups are respectively injected, a live gene electrotransfer instrument is used for carrying out multipoint electric pulse stimulation on an injection part immediately after the injection, and the conditions of the electric pulse stimulation are as follows: voltage 60V, pulse time 50ms, pulse number 1Hz 6 times.

ELISA method for detecting specific antibody in immune mouse serum

At week 2 after the last immunization, each group of immunized mice was bled and serum was isolated. ELISA plates were coated with PSCA, PSMA, PSA and PAP fusion proteins, respectively, to detect the levels of specific antibodies induced following vaccine immunization. By measuring the OD value of each group of serum after ELISA, the result shows that the average of 4 antibodies induced by the vaccine group is higher than that of each control group (figure 6), and the vaccine can induce high-titer antibodies.

Detecting secretion of IFN-gamma specific to splenocytes of immunized mice by ELISPOT method

5.1 isolation of immunized mouse spleen lymphocyte suspensions:

in 2 weeks after the last immunization, each group of immunized mice were killed by neck-breaking, soaked in 75% ethanol for 5min, and then placed in an ultra-clean bench sterilized by ultraviolet irradiation;

taking out the spleen of a mouse by operation under the aseptic condition, shearing the spleen into a 200-mesh cell sieve, putting the cell sieve into a serum-free plate with 1640 culture medium, and grinding the spleen uniformly by using a grinding rod;

gently washing out the lymphocytes in the cell sieve by using a serum-free 1640 culture medium;

adding 5mL of mouse lymphocyte separation liquid into a centrifuge tube, slowly dripping the spleen cell suspension obtained in the step c above the lymphocyte separation liquid along the tube wall (taking care not to mix the two), and centrifuging at the room temperature at 2500rpm for 30 min;

centrifuging, dividing the liquid into three layers, collecting the middle white membrane, adding 10mL serum-free 1640 culture medium, and cleaning lymphocytes for 2 times (1500rpm/min, 10 min); resuspended in 10mL serum-free medium and counted.

5.2 detection of IFN-. gamma.secretion from splenocytes from immunized mice by ELISPOT method

The procedure was carried out according to the Mouse IFN-. gamma.coated ELISPOT kit, Dake Biotech Ltd, as follows:

the first day: inoculating cells, adding stimuli, culturing (strict attention to aseptic operation)

1. Activation of pre-coated plates: adding 200 μ L EZ-CultureTM serum-free culture medium or RPMI-1640 culture medium into each well, standing at room temperature for 5-10min, and deducting.

2. Adding a cell suspension: the adjusted concentration of cell suspension was added to each experimental well at 100. mu.L/well.

Positive control wells: the cell concentration can be 1 × 10⁵cells/well; negative control wells: the cell concentration can be 1 × 10⁵cells/well; background negative control: addingAdding into culture medium for resuspension cells (EZ-CultureTM serum-free culture medium or RPMI-1640 culture medium containing fetal bovine serum); experiment hole: the sample cell concentration is self-adjusted by the experimenter according to the experiment.

3. Adding a stimulus: 10 μ L/well, as follows: positive control wells: adding positive irritant working solution.

Negative control wells [ wells containing background negative control ]: adding EZ-CultureTM serum-free medium (or medium for re-suspending cells); experiment hole: the experimenter's own stimuli (formulated with EZ-CultureTM serum-free medium or RPMI 1640 at 10 Xfinal concentration) were added.

4. And (3) incubation: after all the samples and stimuli were added, the plate cover was closed. Put at 37 ℃ with 5% CO₂Culturing in incubator for 16-20 hr.

The next day: post-culture manipulation (sterile manipulation is no longer required)

1. Cell lysis: pour the cells and media from the wells. Ice-cold deionized water was added, and the cells were hypotonic lysed by placing in a refrigerator at 200. mu.L/well for 10min at 4 ℃.

2. Washing the plate: the well was decanted, washed 5-7 times with 1 × Washing buffer, 200 μ L/well. The residence time is 30-60s each time. And finally, drying the water-absorbing paper in a buckling way.

3. And (3) incubation of the detection antibody: diluted biotin-labeled antibody working solution was added to each assay well at 100. mu.L/well. Incubate at 37 ℃ for 1 hr.

4. Washing the plate: the well was decanted, washed 5 times with 1 × Washing buffer, 200 μ L/well. The residence time is 30-60s each time. And finally, drying the water-absorbing paper in a buckling way.

5. Incubation with enzyme-linked avidin: diluted enzyme-labeled avidin working solution was added to each experimental well at 100. mu.L/well. Incubate at 37 ℃ for 1 hr.

6. Washing the plate: the well was decanted, washed 5 times with 1 × Washing buffer, 200 μ L/well. The residence time is 30-60s each time. And finally, drying the water-absorbing paper in a buckling way.

7. Color development: the AEC color developing solution working solution prepared in situ is added into each experimental hole, and the volume is 100 mu L/well. Standing at room temperature in dark for 15-45min (at 20-25 deg.C, developing for 25min is suitable). If the room temperature is lower than 20 ℃, the color development is recommended to be carried out in an incubator at 37 ℃, and the examination is carried out once every 5-10 min.

8. And (3) stopping color development: the liquid in the wells was poured out, the plate base was uncovered, the front and back sides and base were washed 3-5 times with deionized/tap water, and the development was stopped. The plate is placed in a cool place at room temperature, and the base is closed after the plate is naturally dried.

Spot count on ELISPOT plates and record various parameters of spots for statistical analysis.

Peptides pool of 4 antigens were added to the experimental wells at a concentration of 1. mu.g/well as stimulators, respectively. The results of ELISPOT experiments are shown in fig. 7 and 8: the mice in the immunization group of the vaccine plasmid group can detect the secretion of IFN-gamma of specific lymphocytes, the adjuvant group can detect very low secretion of nonspecific IFN-gamma, and other control groups are negative. Through statistical analysis, the number of spots generated after ELISPOT experiments of different groups of mice stimulated by 4 peptides pool respectively is compared, and the vaccine plasmid group after immunization has significant difference (P is less than 0.001) compared with the normal saline group. The experimental result shows that the vaccine plasmid can induce strong cellular immune response.

The above disclosure is only for the specific embodiment of the present invention, but the present invention is not limited thereto, and any variations that can be made by those skilled in the art should fall within the scope of the present invention.

Sequence listing

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<120> a nucleic acid vaccine for prostate cancer

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gtaaccctgg aaaatgggaa acagctgacc gttaaaagac aaggactcta ttatatctat 2040

gcccaagtca ccttctgttc caatcgggaa gcttcgagtc aagctccatt tatagccagc 2100

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Thr Ile Ser Ala Ala Val Cys Ala Gly Val

1 5 10

<210> 9

<211> 9

<212> PRT

<213> Homo sapiens

<400> 9

Ala Leu Gly Pro Gly Thr Ala Leu Leu

1 5

<210> 10

<211> 9

<212> PRT

<213> Homo sapiens

<400> 10

Ala Ile Leu Ala Leu Leu Pro Ala Leu

1 5

<210> 11

<211> 9

<212> PRT

<213> Homo sapiens

<400> 11

Ala Leu Leu Met Ala Gly Leu Ala Leu

1 5

<210> 12

<211> 10

<212> PRT

<213> Homo sapiens

<400> 12

Leu Leu Cys Thr Ser Cys Leu Ala Gly Val

1 5 10

<210> 13

<211> 9

<212> PRT

<213> Homo sapiens

<400> 13

Leu Leu His Gly Thr Ala Ser Ala Val

1 5

<210> 14

<211> 9

<212> PRT

<213> Homo sapiens

<400> 14

Val Leu Ala Gly Gly Pro Pro Leu Leu

1 5

<210> 15

<211> 10

<212> PRT

<213> Homo sapiens

<400> 15

Leu Leu Gly Gly Ala Gly Val Ala Thr Ile

1 5 10

<210> 16

<211> 198

<212> PRT

<213> Homo sapiens

<400> 16

Thr Leu Met Ser Ala Met Thr Ala Leu Ala Ala Leu Pro Pro Pro Gly

1 5 10 15

Gly Ile Ser Ile Thr Ala Pro Ile Leu Leu Thr Gly Pro Ile Pro Val

20 25 30

His Thr Val Ser Leu Ser Gly Ala Ala Leu Leu Thr Leu Pro Pro Ala

35 40 45

Ala Cys Pro Ala Pro Gly Gly Leu Leu Ser Gly Thr Leu Gly Ser Gly

50 55 60

Gly Pro Leu Leu Ala Leu His Pro Thr Leu Ser Pro Leu Ala Thr Leu

65 70 75 80

Ser Ser Leu Ser Gly Pro Ala Ala Gly Ala Leu Pro Gly Ile Thr Ser

85 90 95

Leu Val Thr Ala Pro Leu Thr Cys Gly Ser Val His Ala Pro Thr Leu

100 105 110

Pro Ser Thr Ala Thr Gly Ala Ala Met Ile Leu Leu Leu Gly Leu Ser

115 120 125

Gly Leu Ser Leu Leu Ser Leu Thr Gly Ile His Leu Gly Leu Gly Leu

130 135 140

Ser Ala Leu Gly Gly Gly Val Leu Val Ala Gly Ile Leu Leu Ala Met

145 150 155 160

Leu Leu Ala Thr Gly Pro Gly Leu Thr Leu Leu Leu Val Met Thr Ser

165 170 175

Ala His Ala Thr Thr Val Ser Gly Leu Gly Met Ala Leu Ala Val Thr

180 185 190

Ala Gly Leu Leu Leu Leu

195

<210> 17

<211> 26

<212> PRT

<213> Homo sapiens

<400> 17

His Leu Thr Pro Leu Leu Leu Gly Cys Val Ala Leu His Ile Ile Ser

1 5 10 15

Ala Ala Val Cys Ala Gly Val Ala Ala Thr

20 25

<210> 18

<211> 65

<212> PRT

<213> Homo sapiens

<400> 18

Leu Leu Cys Thr Ser Cys Leu Ala Gly Val Ser Ala Gly Ala Cys Leu

1 5 10 15

Gly Val Gly Ala Cys Thr Gly Leu Gly Gly Gly Cys Thr Thr Ala Ala

20 25 30

Ile Ala Ala Val Gly Leu Leu Thr Val Ile Ser Leu Gly Cys Ser Leu

35 40 45

Ala Cys Val Ala Ala Ser Gly Ala Thr Thr Val Gly Leu Leu Ala Ile

50 55 60

Gly

65

<210> 19

<211> 32

<212> PRT

<213> Homo sapiens

<400> 19

Leu Leu His Gly Thr Ala Ser Ala Val Ala Thr Ala Ala Ala Pro Ala

1 5 10 15

Thr Leu Cys Ala Gly Ala Leu Val Leu Ala Gly Gly Pro Pro Leu Leu

20 25 30

<210> 20

<211> 4833

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccatcga ttcgaacgat cgttataact agggcggcca attccgcccc 780

tctccctccc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg 840

tttgtctata tgtgattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa 900

cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg 960

caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca 1020

acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc 1080

ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt 1140

gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg 1200

ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca 1260

tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac 1320

gtggttttcc tttgaaaaac acgatgataa gcttgccaca acccgggata attcctgcag 1380

ccaatgcata tggcacccgc ccgctcgccc agccccagca cgcagccctg ggagcatgtg 1440

aatgccatcc aggaggcccg gcgtctcctg aacctgagta gagacactgc tgctgagatg 1500

aatgaaacag tagaagtcat ctcagaaatg tttgacctcc aggagccgac ctgcctacag 1560

acccgcctgg agctgtacaa gcagggcctg cggggcagcc tcaccaagct caagggcccc 1620

ttgaccatga tggccagcca ctacaagcag cactgccctc caaccccgga aacttcctgt 1680

gcaacccaga ctatcacctt tgaaagtttc aaagagaacc tgaaggactt tctgcttgtc 1740

atcccctttg actgctggga gccagtccag gagggcggca ggggtcgacg cggcggcggt 1800

ctttctcact tctgttcagg tgttatccac gtgaccaagg aagtgaaaga agtggcaacg 1860

ctgtcctgtg gtcacaatgt ttctgttgaa gagctggcac aaactcgcat ctactggcaa 1920

aaggagaaga aaatggtgct gactatgatg tctggggaca tgaatatatg gcccgagtac 1980

aagaaccgga ccatctttga tatcactaat aacctctcca ttgtgatcct ggctctgcgc 2040

ccatctgacg agggcacata cgagtgtgtt gttctgaagt atgaaaaaga cgctttcaag 2100

cgggaacacc tggctgaagt gacgttatca gtcaaagctg acttccctac acctagtata 2160

tctgactttg aaattccaac ttctaatatt agaaggataa tttgctcaac ctctggaggt 2220

tttccagagc ctcacctctc ctggttggaa aatggagaag aattaaatgc catcaacaca 2280

acagtttccc aagatcctga aactgagctc tatgctgtta gcagcaaact ggatttcaat 2340

atgacaacca accacagctt catgtgtctc atcaagtatg gacatttaag agtgaatcag 2400

accttcaact ggaatacaac caagcaagag cattttcctg ataacctgct cccatcctgg 2460

gccattacct taatctcagt aaatggaatt tttgtgatat gctgcctgac ctactgcttt 2520

gccccaagat gcagagagag aaggaggaat gagagattga gaagggaaag tgtacgccct 2580

gtataagaat tctgcagata tccagcacag tggcggccgc tcgagtctag agggcccgtt 2640

taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 2700

tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 2760

gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 2820

caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 2880

tctatggctt ctactgggcg gttttatgga cagcaagcga accggaattg ccagctgggg 2940

cgccctctgg taaggttggg aagccctgca aagtaaactg gatggctttc tcgccgccaa 3000

ggatctgatg gcgcagggga tcaagctctg atcaagagac aggatgagga tcgtttcgca 3060

tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg 3120

gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag 3180

cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc 3240

aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc 3300

tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg 3360

atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc 3420

ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca 3480

tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag 3540

agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg 3600

gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg 3660

gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca 3720

tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc 3780

tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg 3840

acgagttctt ctgaattatt aacgcttaca atttcctgat gcggtatttt ctccttacgc 3900

atctgtgcgg tatttcacac cgcatacagg tggcactttt cggggaaatg tgcgcggaac 3960

ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 4020

ctgataaatg cttcaataat agcacgtgct aaaacttcat ttttaattta aaaggatcta 4080

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 4140

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 4200

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 4260

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 4320

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 4380

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 4440

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 4500

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 4560

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 4620

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 4680

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 4740

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 4800

gggcttttgc tggccttttg ctcacatgtt ctt 4833

<210> 21

<211> 10913

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atggcggatg tgtgacatac acgacgccaa aagattttgt tccagctcct gccacctccg 60

ctacgcgaga gattaaccac ccacgatggc cgccaaagtg catgttgata ttgaggctga 120

cagcccattc atcaagtctt tgcagaaggc atttccgtcg ttcgaggtgg agtcattgca 180

ggtcacacca aatgaccatg caaatgccag agcattttcg cacctggcta ccaaattgat 240

cgagcaggag actgacaaag acacactcat cttggatatc ggcagtgcgc cttccaggag 300

aatgatgtct acgcacaaat accactgcgt atgccctatg cgcagcgcag aagaccccga 360

aaggctcgat agctacgcaa agaaactggc agcggcctcc gggaaggtgc tggatagaga 420

gatcgcagga aaaatcaccg acctgcagac cgtcatggct acgccagacg ctgaatctcc 480

taccttttgc ctgcatacag acgtcacgtg tcgtacggca gccgaagtgg ccgtatacca 540

ggacgtgtat gctgtacatg caccaacatc gctgtaccat caggcgatga aaggtgtcag 600

aacggcgtat tggattgggt ttgacaccac cccgtttatg tttgacgcgc tagcaggcgc 660

gtatccaacc tacgccacaa actgggccga cgagcaggtg ttacaggcca ggaacatagg 720

actgtgtgca gcatccttga ctgagggaag actcggcaaa ctgtccattc tccgcaagaa 780

gcaattgaaa ccttgcgaca cagtcatgtt ctcggtagga tctacattgt acactgagag 840

cagaaagcta ctgaggagct ggcacttacc ctccgtattc cacctgaaag gtaaacaatc 900

ctttacctgt aggtgcgata ccatcgtatc atgtgaaggg tacgtagtta agaaaatcac 960

tatgtgcccc ggcctgtacg gtaaaacggt agggtacgcc gtgacgtatc acgcggaggg 1020

attcctagtg tgcaagacca cagacactgt caaaggagaa agagtctcat tccctgtatg 1080

cacctacgtc ccctcaacca tctgtgatca aatgactggc atactagcga ccgacgtcac 1140

accggaggac gcacagaagt tgttagtggg attgaatcag aggatagttg tgaacggaag 1200

aacacagcga aacactaaca cgatgaagaa ctatctgctt ccgattgtgg ccgtcgcatt 1260

tagcaagtgg gcgagggaat acaaggcaga ccttgatgat gaaaaacctc tgggtgtccg 1320

agagaggtca cttacttgct gctgcttgtg ggcatttaaa acgaggaaga tgcacaccat 1380

gtacaagaaa ccagacaccc agacaatagt gaaggtgcct tcagagttta actcgttcgt 1440

catcccgagc ctatggtcta caggcctcgc aatcccagtc agatcacgca ttaagatgct 1500

tttggccaag aagaccaagc gagagttaat acctgttctc gacgcgtcgt cagccaggga 1560

tgctgaacaa gaggagaagg agaggttgga ggccgagctg actagagaag ccttaccacc 1620

cctcgtcccc atcgcgccgg cggagacggg agtcgtcgac gtcgacgttg aagaactaga 1680

gtatcacgca ggtgcagggg tcgtggaaac acctcgcagc gcgttgaaag tcaccgcaca 1740

gccgaacgac gtactactag gaaattacgt agttctgtcc ccgcagaccg tgctcaagag 1800

ctccaagttg gcccccgtgc accctctagc agagcaggtg aaaataataa cacataacgg 1860

gagggccggc ggttaccagg tcgacggata tgacggcagg gtcctactac catgtggatc 1920

ggccattccg gtccctgagt ttcaagcttt gagcgagagc gccactatgg tgtacaacga 1980

aagggagttc gtcaacagga aactatacca tattgccgtt cacggaccgt cgctgaacac 2040

cgacgaggag aactacgaga aagtcagagc tgaaagaact gacgccgagt acgtgttcga 2100

cgtagataaa aaatgctgcg tcaagagaga ggaagcgtcg ggtttggtgt tggtgggaga 2160

gctaaccaac cccccgttcc atgaattcgc ctacgaaggg ctgaagatca ggccgtcggc 2220

accatataag actacagtag taggagtctt tggggttccg ggatcaggca agtctgctat 2280

tattaagagc ctcgtgacca aacacgatct ggtcaccagc ggcaagaagg agaactgcca 2340

ggaaatagtt aacgacgtga agaagcaccg cgggaagggg acaagtaggg aaaacagtga 2400

ctccatcctg ctaaacgggt gtcgtcgtgc cgtggacatc ctatatgtgg acgaggcttt 2460

cgctagccat tccggtactc tgctggccct aattgctctt gttaaacctc ggagcaaagt 2520

ggtgttatgc ggagacccca agcaatgcgg attcttcaat atgatgcagc ttaaggtgaa 2580

cttcaaccac aacatctgca ctgaagtatg tcataaaagt atatccagac gttgcacgcg 2640

tccagtcacg gccatcgtgt ctacgttgca ctacggaggc aagatgcgca cgaccaaccc 2700

gtgcaacaaa cccataatca tagacaccac aggacagacc aagcccaagc caggagacat 2760

cgtgttaaca tgcttccgag gctgggcaaa gcagctgcag ttggactacc gtggacacga 2820

agtcatgaca gcagcagcat ctcagggcct cacccgcaaa ggggtatacg ccgtaaggca 2880

gaaggtgaat gaaaatccct tgtatgcccc tgcgtcggag cacgtgaatg tactgctgac 2940

gcgcactgag gataggctgg tgtggaaaac gctggccggc gatccctgga ttaaggtcct 3000

atcaaacatt ccacagggta actttacggc cacattggaa gaatggcaag aagaacacga 3060

caaaataatg aaggtgattg aaggaccggc tgcgcctgtg gacgcgttcc agaacaaagc 3120

gaacgtgtgt tgggcgaaaa gcctggtgcc tgtcctggac actgccggaa tcagattgac 3180

agcagaggag tggagcacca taattacagc atttaaggag gacagagctt actctccagt 3240

ggtggccttg aatgaaattt gcaccaagta ctatggagtt gacctggaca gtggcctgtt 3300

ttctgccccg aaggtgtccc tgtattacga gaacaaccac tgggataaca gacctggtgg 3360

aaggatgtat ggattcaatg ccgcaacagc tgccaggctg gaagctagac ataccttcct 3420

gaaggggcag tggcatacgg gcaagcaggc agttatcgca gaaagaaaaa tccaaccgct 3480

ttctgtgctg gacaatgtaa ttcctatcaa ccgcaggctg ccgcacgccc tggtggctga 3540

gtacaagacg gttaaaggca gtagggttga gtggctggtc aataaagtaa gagggtacca 3600

cgtcctgctg gtgagtgagt acaacctggc tttgcctcga cgcagggtca cttggttgtc 3660

accgctgaat gtcacaggcg ccgataggtg ctacgaccta agtttaggac tgccggctga 3720

cgccggcagg ttcgacttgg tctttgtgaa cattcacacg gaattcagaa tccaccacta 3780

ccagcagtgt gtcgaccacg ccatgaagct gcagatgctt gggggagatg cgctacgact 3840

gctaaaaccc ggcggcatct tgatgagagc ttacggatac gccgataaaa tcagcgaagc 3900

cgttgtttcc tccttaagca gaaagttctc gtctgcaaga gtgttgcgcc cggattgtgt 3960

caccagcaat acagaagtgt tcttgctgtt ctccaacttt gacaacggaa agagaccctc 4020

tacgctacac cagatgaata ccaagctgag tgccgtgtat gccggagaag ccatgcacac 4080

ggccgggtgt gcaccatcct acagagttaa gagagcagac atagccacgt gcacagaagc 4140

ggctgtggtt aacgcagcta acgcccgtgg aactgtaggg gatggcgtat gcagggccgt 4200

ggcgaagaaa tggccgtcag cctttaaggg agcagcaaca ccagtgggca caattaaaac 4260

agtcatgtgc ggctcgtacc ccgtcatcca cgctgtagcg cctaatttct ctgccacgac 4320

tgaagcggaa ggggaccgcg aattggccgc tgtctaccgg gcagtggccg ccgaagtaaa 4380

cagactgtca ctgagcagcg tagccatccc gctgctgtcc acaggagtgt tcagcggcgg 4440

aagagatagg ctgcagcaat ccctcaacca tctattcaca gcaatggacg ccacggacgc 4500

tgacgtgacc atctactgca gagacaaaag ttgggagaag aaaatccagg aagccattga 4560

catgaggacg gctgtggagt tgctcaatga tgacgtggag ctgaccacag acttggtgag 4620

agtgcacccg gacagcagcc tggtgggtcg taagggctac agtaccactg acgggtcgct 4680

gtactcgtac tttgaaggta cgaaattcaa ccaggctgct attgatatgg cagagatact 4740

gacgttgtgg cccagactgc aagaggcaaa cgaacagata tgcctatacg cgctgggcga 4800

aacaatggac aacatcagat ccaaatgtcc ggtgaacgat tccgattcat caacacctcc 4860

caggacagtg ccctgcctgt gccgctacgc aatgacagca gaacggatcg cccgccttag 4920

gtcacaccaa gttaaaagca tggtggtttg ctcatctttt cccctcccga aataccatgt 4980

agatggggtg cagaaggtaa agtgcgagaa ggttctcctg ttcgacccga cggtaccttc 5040

agtggttagt ccgcggaagt atgccgcatc tacgacggac cactcagatc ggtcgttacg 5100

agggtttgac ttggactgga ccaccgactc gtcttccact gccagcgata ccatgtcgct 5160

acccagtttg cagtcgtgtg acatcgactc gatctacgag ccaatggctc ccatagtagt 5220

gacggctgac gtacaccctg aacccgcagg catcgcggac ctggcggcag atgtgcaccc 5280

tgaacccgca gaccatgtgg acctcgagaa cccgattcct ccaccgcgcc cgaagagagc 5340

tgcatacctt gcctcccgcg cggcggagcg accggtgccg gcgccgagaa agccgacgcc 5400

tgccccaagg actgcgttta ggaacaagct gcctttgacg ttcggcgact ttgacgagca 5460

cgaggtcgat gcgttggcct ccgggattac tttcggagac ttcgacgacg tcctgcgact 5520

aggccgcgcg ggtgcatata ttttctcctc ggacactggc agcggacatt tacaacaaaa 5580

atccgttagg cagcacaatc tccagtgcgc acaactggat gcggtccagg aggagaaaat 5640

gtacccgcca aaattggata ctgagaggga gaagctgttg ctgctgaaaa tgcagatgca 5700

cccatcggag gctaataaga gtcgatacca gtctcgcaaa gtggagaaca tgaaagccac 5760

ggtggtggac aggctcacat cgggggccag attgtacacg ggagcggacg taggccgcat 5820

accaacatac gcggttcggt acccccgccc cgtgtactcc cctaccgtga tcgaaagatt 5880

ctcaagcccc gatgtagcaa tcgcagcgtg caacgaatac ctatccagaa attacccaac 5940

agtggcgtcg taccagataa cagatgaata cgacgcatac ttggacatgg ttgacgggtc 6000

ggatagttgc ttggacagag cgacattctg cccggcgaag ctccggtgct acccgaaaca 6060

tcatgcgtac caccagccga ctgtacgcag tgccgtcccg tcaccctttc agaacacact 6120

acagaacgtg ctagcggccg ccaccaagag aaactgcaac gtcacgcaaa tgcgagaact 6180

acccaccatg gactcggcag tgttcaacgt ggagtgcttc aagcgctatg cctgctccgg 6240

agaatattgg gaagaatatg ctaaacaacc tatccggata accactgaga acatcactac 6300

ctatgtgacc aaattgaaag gcccgaaagc tgctgccttg ttcgctaaga cccacaactt 6360

ggttccgctg caggaggttc ccatggacag attcacggtc gacatgaaac gagatgtcaa 6420

agtcactcca gggacgaaac acacagagga aagacccaaa gtccaggtaa ttcaagcagc 6480

ggagccattg gcgaccgctt acctgtgcgg catccacagg gaattagtaa ggagactaaa 6540

tgctgtgtta cgccctaacg tgcacacatt gtttgatatg tcggccgaag actttgacgc 6600

gatcatcgcc tctcacttcc acccaggaga cccggttcta gagacggaca ttgcatcatt 6660

cgacaaaagc caggacgact ccttggctct tacaggttta atgatcctcg aagatctagg 6720

ggtggatcag tacctgctgg acttgatcga ggcagccttt ggggaaatat ccagctgtca 6780

cctaccaact ggcacgcgct tcaagttcgg agctatgatg aaatcgggca tgtttctgac 6840

tttgtttatt aacactgttt tgaacatcac catagcaagc agggtactgg agcagagact 6900

cactgactcc gcctgtgcgg ccttcatcgg cgacgacaac atcgttcacg gagtgatctc 6960

cgacaagctg atggcggaga ggtgcgcgtc gtgggtcaac atggaggtga agatcattga 7020

cgctgtcatg ggcgaaaaac ccccatattt ttgtggggga ttcatagttt ttgacagcgt 7080

cacacagacc gcctgccgtg tttcagaccc acttaagcgc ctgttcaagt tgggtaagcc 7140

gctaacagct gaagacaagc aggacgaaga caggcgacga gcactgagtg acgaggttag 7200

caagtggttc cggacaggct tgggggccga actggaggtg gcactaacat ctaggtatga 7260

ggtagagggc tgcaaaagta tcctcatagc catggccacc ttggcgaggg acattaaggc 7320

gtttaagaaa ttgagaggac ctgttataca cctctacggc ggtcctagat tggtgcgtta 7380

atacacagaa ttctgattgg atcccgggta attaattgaa ttacatccct acgcaaacgt 7440

tttacggccg ccggtggcgc ccgcgcccgg cggcccgtcc ttggccgttg caggccactc 7500

cggtggctcc cgtcgtcccc gacttccagg cccagcagat gcagcaactc atcagcgccg 7560

taaatgcgct gacaatgaga cagaacgcaa ttgctcctgc taggcctccc aaaccaaaga 7620

agaagaagac aaccaaacca aagccgaaaa cgcagcccaa gaagatcaac ggaaaaacgc 7680

agcagcaaaa gaagaaagac aagcaagccg acaagaagaa gaagaaaccc ggaaaaagag 7740

aaagaatgtg catgaagatt gaaaatgact gtatcttcgt atgcggctag ccacagtaac 7800

gtagtgtttc cagacatgtc gggcaccgca ctatcatggg tgcagaaaat ctcgggtggt 7860

ctgggggcct tcgcaatcgg cgctatcctg gtgctggttg tggtcacttg cattgggctc 7920

cgcagataag ttagggtagg caatggcatt gatatagcaa gaaaattgaa aacagaaaaa 7980

gttagggtaa gcaatggcat ataaccataa ctgtataact tgtaacaaag cgcaacaaga 8040

cctgcgcaat tggccccgtg gtccgcctca cggaaactcg gggcaactca tattgacaca 8100

ttaattggca ataattggaa gcttacataa gcttaattcg acgaataatt ggatttttat 8160

tttattttgc aattggtttt taataaaaaa aaaaaaaaaa aaaaaaacta gagatcataa 8220

tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 8280

tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 8340

atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 8400

attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctagtctg 8460

cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 8520

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 8580

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 8640

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 8700

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 8760

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 8820

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 8880

ctttctcaat gctcgcgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 8940

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 9000

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 9060

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 9120

ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 9180

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 9240

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 9300

tctacggggc attctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 9360

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 9420

atctaaagta tatatgagta aacttggtct gacagactag tatgattgaa caagatggat 9480

tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac 9540

agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc 9600

tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc 9660

tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag 9720

cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc 9780

ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg 9840

atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc 9900

ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc 9960

cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga 10020

cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca 10080

tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg 10140

atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg 10200

ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgactcg 10260

aggcatgcaa gctgtaatca attacggggt cattagttca tagcccatat atggagttcc 10320

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 10380

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 10440

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 10500

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 10560

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 10620

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 10680

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 10740

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 10800

tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttaac 10860

tggcttatcg aaattaatac gactcactat agggagaccg gaagcttgaa ttc 10913

Claims

1. A prostate cancer nucleic acid vaccine is characterized in that the nucleic acid vaccine is formed by fusing gene sequences of a region which can induce CD8+ T cell immune response and covers HLA-A2 epitope and expresses prostate cancer antigens PSCA, PAP, PSMA and PSA, a gene sequence which expresses a signal peptide of FLT3L and an extracellular region is fused at the upstream of the nucleic acid vaccine, and a gene sequence which expresses CD40L is fused at the downstream of the nucleic acid vaccine;

2. The prostate cancer nucleic acid vaccine of claim 1, wherein the gene sequence of the prostate cancer nucleic acid vaccine is represented by the nucleotide sequence table SEQ ID NO 1.

3. A plasmid vector comprising the prostate cancer nucleic acid vaccine of claim 1 or 2.

4. The plasmid vector comprising a prostate cancer nucleic acid vaccine according to claim 3, wherein the original plasmid vector of the plasmid vector is pSVK 1.

5. Use of the prostate cancer nucleic acid vaccine of claim 1 or 2 for the manufacture of a medicament against prostate cancer.

6. Use of the prostate cancer nucleic acid vaccine of claim 5, for the manufacture of a medicament against prostate cancer, comprising the prostate cancer nucleic acid vaccine of claim 1 or 2 and a co-stimulatory adjuvant comprising a cytokine, a chemokine and a bacterial toxin.

7. Use of the prostate cancer nucleic acid vaccine of claim 6, in the manufacture of a medicament against prostate cancer, wherein the cytokines are the cytokines GM-CSF and B7.1.